The highly conserved SNF1/AMP-activated protein kinase (AMPK) is a central energy regulator in eukaryotes. In humans, AMPK is implicated in the pathogenesis of disease, including type 2 diabetes, obesity and cancer, and AMPK is a target of anti-diabetic drugs. The yeast SNF1 pathway has provided a paradigm for understanding the human pathway. We propose to continue our functional analysis of the SNF1/AMPK pathway in the yeast system, which offers the advantages of powerful genetics. SNF1/AMPK, a heterotrimer, is activated by phosphorylation of the activation loop on the catalytic subunit in response to metabolic and other stress. Accumulating evidence points to the importance of protein phosphatases in regulating SNF1/AMPK, but the phosphatases that are involved and the mechanisms regulating dephosphorylation are not well understood. We propose studies to elucidate the roles of phosphatases in the SNF1 pathway. We recently found that, in addition to Reg1-Glc7 protein phosphatase 1 (PP1), type 2A-like phosphatase Sit4 has a role in the SNF1 pathway. We propose genetic and biochemical approaches to determine whether these two phosphatases together are responsible for dephosphorylation of SNF1 during growth on high levels of glucose and to address their roles in different stress responses. The interactions of these phosphatases with SNF1 will be investigated under different conditions. The roles of the three SNF1 subunits in determining binding specificity for each phosphatase will be assessed, and various alterations of SNF1 that prevent dephosphorylation of the activation loop will be tested for effects on interactions with phosphatases. The proposed studies should provide insight into mechanisms that control phosphatase function under different conditions. Finally, it will be important to identify all the major phosphatases that regulate the SNF1 pathway. Evidence suggests Ptc1 as a promising candidate, and we plan to assess its role in regulation of SNF1. We will also assess the roles of several other candidates, and, if appropriate, undertake a search for other phosphatases. The proposed studies will provide new insights into regulatory mechanisms of general relevance for SNF1/AMPK pathways and will inform efforts to identify human phosphatases for AMPK. We further anticipate that this work will point to new strategies for preventive interventions for metabolic disorders and cancer, by targeting phosphatases and their interactions with AMPK to activate the kinase.